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Ligand recognition and G-protein coupling of trace amine receptor TAAR1

Nature volume 624, pages 672–681 (2023)Cite this article

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Abstract

Trace-amine-associated receptors (TAARs), a group of biogenic amine receptors, have essential roles in neurological and metabolic homeostasis1. They recognize diverse endogenous trace amines and subsequently activate a range of G-protein-subtype signalling pathways2,3. Notably, TAAR1 has emerged as a promising therapeutic target for treating psychiatric disorders4,5. However, the molecular mechanisms underlying its ability to recognize different ligands remain largely unclear. Here we present nine cryo-electron microscopy structures, with eight showing human and mouse TAAR1 in a complex with an array of ligands, including the endogenous 3-iodothyronamine, two antipsychotic agents, the psychoactive drug amphetamine and two identified catecholamine agonists, and one showing 5-HT1AR in a complex with an antipsychotic agent. These structures reveal a rigid consensus binding motif in TAAR1 that binds to endogenous trace amine stimuli and two extended binding pockets that accommodate diverse chemotypes. Combined with mutational analysis, functional assays and molecular dynamic simulations, we elucidate the structural basis of drug polypharmacology and identify the species-specific differences between human and mouse TAAR1. Our study provides insights into the mechanism of ligand recognition and G-protein selectivity by TAAR1, which may help in the discovery of ligands or therapeutic strategies for neurological and metabolic disorders.

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Fig. 1: Activation of TAAR1 and downstream G-protein subtypes.
Fig. 2: The overall structure of TAAR1–Gs complexes and the 5-HT1AR–Gi1 complex.
Fig. 3: Recognition mechanism of the endogenous amine T1AM by TAAR1.
Fig. 4: Recognition of SEP-363856 and RO-6889450 by TAAR1 and 5-HT1AR.
Fig. 5: Recognition of the (S)-AMPH isomer by TAAR1.

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Data availability

Cryo-EM coordinates and density maps have been deposited at the PDB and the Electron Microscopy Data Bank under accession codes 8JLN, EMD-36401 (T1AM–hTAAR1–Gs complex); 8JLO, EMD-36402 (SEP-363856–hTAAR1–Gs complex); 8JLP, EMD-36403 (RO-6889450–hTAAR1–Gs complex); 8JLQ, EMD-36404 (fenoldopam–hTAAR1–Gs complex); 8JLR, EMD-36405 (A77636–hTAAR1–Gs complex); 8JLJ, EMD-36399 (T1AM–mTAAR1–Gs complex); 8JLK, EMD-36400 (SEP-363856–mTAAR1–Gs complex); 8JSO, EMD-36625 ((S)-AMPH–hTAAR1–Gs complex); and 8JSP, EMD-36626 (SEP-363856–5-HT1AR–Gi complex).

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Acknowledgements

We thank the staff at the Translational Medicine Core Facility of Advanced Medical Research Institute at Shandong University for their technical assistance; the staff of Sichuan University West China Cryo-EM Center and Cryo-EM Center in Southern University of Science and Technology for cryo-EM data collection; and the staff at the Cryo-EM Center in Shandong University for technical support. This work was supported by the National Natural Science Foundation of China (32100988; 32371288 to W.Y., T2221004 to S.Y. and Z.S., 31972916 to Z.S., 81825022 to J.-P.S., 82271190 and 32100965 to L.C.), Science and Technology Department of Sichuan Province (2022ZYD0085 to Z.S.), Frontiers Medical Center, Tianfu Jincheng Laboratory Foundation (TFJC2023010010 to Z.S.), the China Postdoctoral Science Foundation (BX20220219 to Z.X.), 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (ZYYC20023 to Z.S.), National Science Fund for Excellent Young Scholars (82122070 to F.Y.), National Key R&D Program of China (2022YFC2702603 to F.Y.), STI2030-Major Projects (2021ZD0203000 and 2021ZD0203003 to X.W.), the Beijing National Laboratory for Molecular Sciences (BNLMS202108 to X.W.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB0450102 to X.W.) and the Chinese Academy of Sciences (CAS) Pioneer Hundred Talents Program. This work used resources from the Duyu High Performance Computing Center, Sichuan University, and Big Data Platform at West China Hospital of Sichuan University (WCH-BDP).

Author information

Author notes

  1. These authors contributed equally: Zheng Xu, Lulu Guo, Jingjing Yu, Siyuan Shen, Chao Wu, Weifeng Zhang, Chang Zhao

Authors and Affiliations

  1. Division of Nephrology and Kidney Research Institute, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China

    Zheng Xu, Jingjing Yu, Siyuan Shen, Chao Wu, Chang Zhao, Yue Deng, Xiaowen Tian, Yuying Feng, Hanlin Hou, Lantian Su, Shuo Guo, Heli Wang, Kexin Wang, Peipei Chen, Jie Zhao, Xiaoyu Zhang, Xihao Yong, Shengyong Yang, Wei Yan & Zhenhua Shao

  2. Frontiers Medical Center, Tianfu Jincheng Laboratory, Chengdu, China

    Zheng Xu, Jie Zhao & Zhenhua Shao

  3. Advanced Medical Research Institute, Meili Lake Translational Research Park, Cheeloo College of Medicine, Shandong University, Jinan, China

    Lulu Guo, Fan Yang & Jin-Peng Sun

  4. Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Shandong University, Jinan, China

    Weifeng Zhang & Xiao Yu

  5. Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China

    Hongshuang Wang & Xiaohui Wang

  6. Department of Otolaryngology Head and Neck Surgery, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, China

    Lin Cheng

  7. Department of Thoracic Surgery, West China Hospital, Sichuan University, Chengdu, China

    Lunxu Liu

  8. Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University, Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, China

    Fan Yang & Jin-Peng Sun

  9. School of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei, China

    Xiaohui Wang

  10. Beijing National Laboratory for Molecular Sciences, Beijing, China

    Xiaohui Wang

  11. Department of General Surgery, Qilu Hospital of Shandong University, Jinan, China

    Yunfei Xu

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  1. Zheng Xu
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Contributions

Z.S., W.Y., X. Yu and J.-P.S. initiated structural studies of TAAR and their ligands. Z.X., J.Y., S.S. and Y.F. designed the expression constructs, and purified and prepared the hTAAR1–Gs complexes (T1AM–hTAAR1–Gs, SEP-363856–hTAAR1–Gs, (S)-AMPH–hTAAR1–Gs, RO-6889450–hTAAR1–Gs, fenoldopam–hTAAR1–Gs, A77636–hTAAR1–Gs) and SEP-363856–5-HT1AR–Gi complexes with assistance from H.H. and L.S.; L.G. and W.Z. performed purification of the mTAAR1–Gs complexes (SEP-363856–mTAAR1–Gs, T1AM–mTAAR–Gs) under the supervision of X. Yong and F.Y.; Z.X., C.Z. and X.T. performed cryo-EM screening, data collection, model building, refinement and MD simulations in the study. Hongshuang Wang and X.W. prepared the AMPH. H.H. and L.S. prepared plasmids. C.W., Y.D. and K.W. performed functional assays on T1AM, SEP-363856, RO-6889450, fenoldopam and A77636 with the help of P.C., S.G. and Heli Wang. K.W., S.G. and C.W. contributed to the G-protein-dissociation assay. W.Z. performed radioligand-binding assays. X.Z. contributed to the purification of scFv16. Z.X., S.S., X. Yong and C.W. prepared figures with assistance from X.T., C.Z., L.C., J.Z., L.G. and F.Y.; Z.X., S.S. and C.W. planned and coordinated the entire project with the help of Y.X., X. Yong and L.L.; W.Y. and Z.S. supervised the overall project and wrote the manuscript with the help of J.-P.S., X. Yu, S.Y. and L.L.

Corresponding authors

Correspondence to Xiao Yu, Yunfei Xu, Jin-Peng Sun, Wei Yan or Zhenhua Shao.

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Competing interests

The authors declare no competing interests.

Peer review

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Nature thanks Tao Che, Thomas Keck and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Activation of TAAR1 signalling induced by different ligands.

Concentration dependent activation of hTAAR1 (a, c) and mTAAR1 (b, d) in response to different agonists stimulation examined by G-proteins dissociation assays. Data are presented as mean ± SEM of three independent experiments performed in triplicate.

Extended Data Fig. 2 Structural comparison of TAAR1 with other aminergic receptors.

a, Superposed structures of diverse agonist-bound TAAR1 with T1AM-hTAAR1. The root mean square deviation (RMSD) levels were calculated using T1AM-hTAAR1 structure as the reference. b, Structural superposition of T1AM-hTAAR1 structure with DRD1 (PDB:7CKZ), 5-HT4R (PDB:7XTA), β1AR (PDB:7BTS) and β2AR (PDB:3SN6), respectively. c, Structural comparison of T1AM-hTAAR1 with SEP-363856-hTAAR1 and RO-6889450-hTAAR1 (left panel) or T1AM-mTARR1 with SEP-363856-mTARR1 (right panel) revealed a similar ECL2 in TAAR1 receptor. d, Structural superposition of T1AM-hTAAR1 structure with SEP-363856-bound 5-HT1AR, dopamine-bound DRD1 (PDB:7CKZ) and adrenaline-bound β2AR (PDB: 3SN6).

Extended Data Fig. 3 Pharmacological characterization of T1AM at TAAR1.

a, Sequence alignment of residues involved in T1AM binding in hTAAR1 and mTAAR1. The conserved residues are shown as green, residues that specifically interacted with T1AM are coloured as grey. b, Structural comparison of ligand binding mode of T1AM in mTAAR1 with dopamine in DRD1. c, Heatmap visualizes the effect of mutations in the ligand binding pocket of mTAAR1 in response to T1AM, as determined by the CAMYEL assay. The heatmap is coloured according to the value of ΔpEC50 (ΔpEC50 = pEC50 of mutant - pEC50 of WT) and Emax (relative WT%). ND, not detected or cannot be established over the tested concentration range. Data are presented as mean ± SEM of three independent experiments performed in triplicate. WT, wild type. d, e, Representative curve of key residues involved in activation of hTAAR1 (d) or mTAAR1 (e) by CAMYEL assay. Data represent mean ± SEM from three independent experiments performed in triplicate. WT, wild type. f, Competitive binding curves of T1AM to wild type hTAAR1 or its mutants in pocket 1 and pocket 2. [3H]-tyramine was used in our study. The Ki values of wild type hTAAR1 for T1AM are 23.59 ± 0.62 nM. Data are presented as mean ± SEM of three independent experiments performed in triplicate. g, Representative curve for effects of mutations in pocket 1 (left panel) and pocket 2 (right panel) from hTAAR1 in response to the T1AM stimulation by FlAsH-BRET assay. Values are means ± SEM from three independent experiments performed in triplicate. h, Structural comparison of the hTAAR1-T1AM (sea green) with mTAAR1-T1AM (light salmon) reveals notable differences (hThr/mAla5.42) in ligand binding pocket. Residue are shown as tan sticks in hTAAR1 and medium slate blue sticks in mTAAR1.

Extended Data Fig. 4 Sequence alignment of key residues of TAARs in the ligand binding pocket.

The key residues in pocket 1, pocket 2, pocket 3 and pocket 4 of TAAR1 are sequentially aligned among members of TAAR subfamily.

Extended Data Fig. 5 Pharmacological characterization of SEP-363856 at TAAR1 and 5-HT1AR.

a, 2D representation of interaction of SEP-363856 with h/mTAAR1. b, The superimposed structures of SEP-363856-bound hTAAR1 (green/blue) with SEP-363856-bound mTAAR1 (yellow/cyan) reveal nearly identical binding pose of ligand. c, d, Comparison of the binding mode of SEP-363856 and T1AM in hTAAR1 (c) and mTAAR1 (d). e-g, Glosensor assay of receptors WT and mutations in hTAAR1 (e), mTAAR1 (f) and 5-HT1AR (g) on SEP-363856-induced Gs signalling. Data represent mean ± SEM from three independent experiments performed in triplicate. WT, wild type. h, Competitive binding curves of SEP-363856 to wild type hTAAR1 or its mutants in pocket 1 and pocket 2. [3H]-tyramine was used in our study. The Ki values of wild type hTAAR1 for SEP-363856 are 22.59 ± 0.60 nM. Data are presented as mean ± SEM of three independent experiments performed in triplicate. i, j, Representative curve for effects of the Phe186ECL2 mutations in hTAAR1 (i) or Phe185ECL2 mutations in mTAAR1 (j) on SEP-363856-induced Gs signalling detected by Glosensor assay. Data are presented as mean ± SEM of three independent experiments performed in triplicate. WT, wild type. k, The residues in ECL2 of TAAR1 are sequentially aligned among aminergic receptors. * Representing the corresponding residues in aminergic receptors that relatively to Phe186ECL2 of hTAAR1. l, Structural comparison of the hTAAR1-SEP363856 with 5-HT1AR-SEP363856 reveals that the residues Phe186ECL2 in hTAAR1 and Lys191ECL2 in 5-HT1AR are different. Residues are shown as dodger blue sticks in hTAAR1 and pale violet red sticks in 5-HT1AR, respectively. m, Representative curve for effects of the Lys191ECL2A mutation in 5-HT1AR on SEP-363856-induced Gi signalling detected by Glosensor assay. Data are presented as mean ± SEM of three independent experiments performed in triplicate. WT, wild type.

Extended Data Fig. 6 Structural features of RO-6889450 and (S)-AMPH bound.

a, 2D representation of interaction of RO-6889450 with hTAAR1. b, Representative dose response curves of the RO-6889450-induced FlAsH-BRET ratio for the mutants of residues Phe1544.56 and Ser1945.42 in pocket 3. Values are means ± SEM from three independent experiments performed in triplicate. c, Mutational effects of Phe1544.56A and Ser1945.42A on hTAAR1-Gs (left panel), hTAAR1-Gi (middle panel) or hTAAR1-Gq (right panel) signalling in response to RO-6889450 stimulation by Gαs-γ1, Gαi1-γ2 or Gαq-γ9 dissociation assay. Data are presented as mean ± SEM of three independent experiments performed in triplicate. WT, wild type. d, Structural comparison of (S)-AMPH with SEP-363856-bound in hTAAR1 at pocket 1 and pocket 2. e, Conformational changes of key residues from pocket 2 in TAAR1 binds to (S)-AMPH or T1AM. f, The orientation of (S)-AMPH in 500 ns molecular dynamic (MD) simulation course. g, Heatmap was visualized the effects of mutations of key residues from hTAAR1 in response to (S)-AMPH. The heatmap is coloured according to the value of ΔpEC50 (ΔpEC50 = pEC50 of mutant - pEC50 of WT) and Emax (relative WT%). ND, not detected or cannot be established over the tested concentration range. Data are presented as mean ± SEM of three independent experiments performed in triplicate. WT, wild type. h, Representative curve for effects of mutations in pocket 1 (upper panel) and pocket 2 (lower panel) on (S)-AMPH-induced Gs signalling using Gαs-γ1 dissociation assay. Data are presented as mean ± SEM of three independent experiments performed in triplicate. WT, wild type.

Extended Data Fig. 7 Structural features of fenoldopam- or A77636-bound hTAAR1.

a, Screening of 22 synthetic molecules for hTAAR1 activation, as assessed using the Glosensor assay. At the administration of 10 μM, two of the 22 compounds showed >50% of activity of agonist β-PEA and SEP-363856. Data shown as mean ± standard error of the mean (SEM) (n = 4 biologically independent samples). ***P < 0.001. Comparison between buffer and several ligands. All the data were determined by one-way of variance ANOVA with Dunnett’s test (From left to right, P = < 0.001, <0.001, <0.001, <0.001, 0.999, 0.265, > 0.999, > 0.999, 0.988, > 0.999, 0.999, 0.995, 0.999, 0.971, 0.969, > 0.999, 0.994, 0.994, > 0.999, > 0.999, > 0.999, 0.988, 0.948, > 0.999). b, Representative dose-response curves for hTAAR1-Gs signalling simulated by the endogenous agonist β-PEA, as well as SEP-363856, fenoldopam, and A77636, as determined by the CAMYEL assay. All curves are mean ± SEM of three independent experiments performed in triplicate (n = 3). c, d, Activation potency and efficacy of hTAAR1 and mTAAR1 induced by β-PEA, fenoldopam and A77636 using CAMYEL assay. Data are presented as mean ± SEM of three independent experiments performed in triplicate. e, f, Structural details of fenoldopam (magenta, stick) (e) and A77636 (orange, stick) (f) bound to hTAAR1. Schematic representation of residues contacts with fenoldopam and A77636 in hTAAR1. Residues in pocket 1, 2 and 4 are shown in pink boxes, green boxes and medium slate blue boxes, respectively. Residues located concurrently in pocket 2 and pocket 4 are placed in a green/medium slate blue mixed box. g, Different binding pose of fenoldopam in the structure between DRD1 and hTAAR1. h, Structural comparison of fenoldopam-DRD1 with A77636-hTAAR1.

Extended Data Fig. 8 The role of pocket 4 in TAAR1.

a-h, Structural representations of the polar interaction network of the residues at position 2.61, 2.64, 3.32 and 7.43, for the hTAAR1 with SEP-363856 (a), T1AM (b), (S)-AMPH (c), RO-6889450 (d), fenoldopam (g) and A77636 (h), or mTAAR1 with SEP-363856 (e), T1AM (f), respectively. The estimated hydrogen bonds are represented with red dash lines. i, Representative curve for effects of the S802.61A, R832.64A and H993.28A mutations in hTAAR1 on ligands (fenoldopam, A77636, T1AM, SEP-363856) induced Gs signalling using CAMYEL assay. Data are presented as mean ± SEM of three independent experiments performed in triplicate. WT, wild type. j, Representative curve for effects of the S802.61A, R832.64A and H993.28A mutations in hTAAR1 on ligands (fenoldopam, A77636, T1AM, SEP-363856) binding using FlAsH-BRET assay. Data are presented as mean ± SEM of three independent experiments performed in triplicate.

Extended Data Fig. 9 Activation of TAAR1 by diverse agonists.

a, Cut-away view of the ligand-binding pocket in the hTAAR1 complexes: T1AM, SEP-363856, (S)-AMPH, RO-6889450, fenoldopam and A77636. b, Cut-away view of the ligand-binding pocket in the mTAAR1 complexes: T1AM, SEP-363856. c, The residues of the core ligand binding pocket are resembled in the SEP-363856-, RO-6889450- and fenoldopam-bound structures. The hydrogen bonds shown as red dashed lines. d, e, Structural representation of the detailed interactions of SEP-363856 and RO-6889450 with the key residues in pocket 3 (e), and SEP-363856, A77636 and fenoldopam with the key residues in pocket 4 (f). The hydrogen bonding is shown as red dashed lines. The red arrows indicate direction of movement. f, Structural superimposition of A77636-bound hTAAR1 with SEP-33856-bound mTAAR1 revealed that Pro183ECL2 and Tyr2877.39 in mTAAR1 are different from the allelic residue in hTAAR1. g, Representative curve for effects of the V184ECL2A and V184ECL2P mutations in hTAAR1 on A77636-induced Gs signalling using CAMYEL assay. Data are presented as mean ± SEM of three independent experiments performed in triplicate. WT, wild type. h, j, Schematic diagram representing the key residues that contribute to the Gs (h) or Gi (j) proteins coupling with TAAR1. hTAAR1 is depicted as a green cartoon. Residues crucial for Gs or Gi activation are denoted with blue circle (h) or pink circle (j), respectively. i, k, Heatmap illustrating the effect of mutations on the signalling pathways of hTAAR1-Gs (i) and hTAAR1-Gi (k) in response to (S)-AMPH, as evaluated by the Gαs-γ1 and Gαi1-γ2 dissociation assays. Data are presented as mean ± SEM of three independent experiments performed in double.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics of G protein coupled GPCR complexes
Full size table

Supplementary information

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Supplementary Figs. 1–19, Supplementary Tables 1–5 and Supplementary Methods.

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Xu, Z., Guo, L., Yu, J. et al. Ligand recognition and G-protein coupling of trace amine receptor TAAR1. Nature 624, 672–681 (2023). https://doi.org/10.1038/s41586-023-06804-z

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